Preliminary Research On Viral Like Particle Vaccine And Hydrogen Peroxide Inactivated Vaccine Of Japanese Encephalitis

The pathogen of porcine Japanese encephalitis is Japanese encephalitis virus (JEV),belonging to the flaviviridae family,flavivirus which include Yellow fever virus、JEV、West nile virus and Degue virus.Those Seriously harm to health of human and animal.So far vaccination and killing mosquito are the main measures of prevention and control of JE.This study preliminarily explored a Japanese encephalitis virus particles vaccine,and a new inactivated vaccine.HBC is Hepatitis B core antigen,which can be expressed in a variety of expression systems and self-assemble into virus-like particles.It is a good immunogenic epitope-display carrier.And as reported a new virus inactivating agent-hydrogen peroxide can quickly and effectively inactivate virus,with minimal damage to antigenic structure or immunogenicity in constract with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone.In the experiment a preliminary study of porcine encephalitis inactivated vaccine with hydrogen peroxide as inactivated agent.1Preparation and immunology research of virus-like particles displayed JEV EDⅢ loop3epitopeJEV E protein domain III (EDⅢ) is important region of induction neutralizing antibodies. It was reported that experiments in vitro showed one of cyclic peptide of EDIII(loop3) could interfere with viral adsorption cells and inhibit viral infection.Challenge experiments in mice after inoculation of the protein showed loop3was the strongest protection polypeptide. Therefore, loop3has the potention for new anti-virus vaccines. However loop3epitope peptide was a tiny amino acid sequence which has9amino acid residues.Hepatitis B core protein is a major structural protein of hepatitis B virus, which in a variety of expression systems can be efficiently expressed,and automatically assembled into virus-like particles.Insertion exogenous peptides into particular sites of HBC does not affect the conformation of HBC and self-assembling and the peptides can also be displayed on the surface of the virus-like particles, which can greatly enhance the immunogenicity of epitope peptides. So the recombinant of HBC gene and loop3gene were constructed.Based on the particulate matter as immunogenic carrier, loop3epitope immunogenity was improved. According on the above,this paper constructed the fusion expression plasmid pET28a-HBc-loop3based on the HBC as carrier.JEV EDIII loop3epitope gene was inserted into the MIR region of HBC.SDS-PAGE proved that IPTG induced E.coli high expression of the target protein in supernatant named HBC-loop3. By density gradient centrifugation and gel filtration high purity protein was obtained.TEM observations confirmed that the fusion protein could be self-assembled into virus-like particles.Immunized five five-weeks old mice with the purified HBC-loop3,in peritoneal cavity inoculation,then collected the serum and used a variety of methods to detect specific antibodies,such as ELISA method, hemagglutination inhibition and plaque neutralization test.The results showed tha:antibodies ELISA value (0.59) of protein-immunized group was significantly greater than the value of the PBS group (0.18); protein-immunized group serum were better than the PBS group in hemagglutination inhibition effect; however plaque inhibition tests showed that neutralizing antibodies was not detected in the serum.2Preliminary trial and immunization of swine Japanese encephalitis virus inactivating agent based hydrogen peroxide as inactivated reagentsIn this study we used immunogenic swine Japanese encephalitis virus-NANJING08strain.In the experiments we chose hydrogen peroxide and formaldehyde to inactivate JEV virus,then inoculated five five-weeks old mice in peritoneal cavity inoculation,1×106PFU per mouse,then using ELISA,hemagglutination inhibition and plaque neutralization test to evaluate the effect of immunization.And the result of ELISA revealed that virus inactivated by H2O2could produce higher level of ELISA value (1.20) than formaldehyde (1.10);HI test revealed that virus antigen inactivated by H2O2induced higher HI value;at last plaque neutralization test confirmed that virus antigen inactivated by H2O2could produce more neutralizing antibodies.Preliminary results showed that hydrogen peroxide could effectively inactivate Japanese encephalitis virus, and inactivated vaccine of Japanese encephalitis by hydrogen peroxide could induce higher protective antibody levels, so hydrogen peroxide is a very promising reageant inactivated vaccine Japanese encephalitis.